Tierärzt. Hochsch., Diss., 2003--Hannover.

Avian intestinal spirochaetosis (AIS) is a relatively recently recognized disease of commercial layer and meat breeder chickens resulting from colonization of the gastrointestinal tract by anaerobic spirochaetal bacteria of the genus Brachyspira. AIS is characterised by delayed and/or reduced egg production and chronic diarrhoea. This thesis describes an investigation into the prevalence, pathogenicity and control of species of avian intestinal spirochaete in Australia. Faeces samples from chickens in 22 flocks of laying hens, 19 broiler flocks and 28 breeder flocks in Queensland, New South Wales, Victoria, Tasmania and South Australia were subjected to selective anaerobic culture for Brachyspira species. Spirochaete isolates then were speciated using phenotypic characteristics and specific polymerase chain reaction amplifications. A highly significant association was found between colonisation with Brachyspira species and the occurrence of wet litter and/or reduced production in both broiler breeder and layer flocks in eastern Australia. Multilocus enzyme electrophoresis (MLEE) then was used to help confirm the identity of the spirochaetes, and to examine their genetic relationships and disease associations. MLEE divided the isolates into five known Brachyspira species groups: Brachyspira murdochii, B. intermedia, B. pilosicoli, B. innocens, and “B. pulli”. Three new MLEE groups each containing single isolates were also identified. All farms with production problems or wet litter were colonised with the pathogenic species, B. intermedia and/or B. pilosicoli. The pathogenic potential of single isolates of B. pilosicoli and B. innocens then were experimentally evaluated in adult broiler breeders, confirming that infection with B. pilosicoli can result in serious egg production losses, whilst B. innocens is non-pathogenic. Antimicrobial susceptibility testing showed that the pathogenic spirochaetes B. pilosicoli and B. intermedia were both susceptible to tiamulin, lincomycin, metronidazole and tetracycline, while a lack of susceptibility to tylosin was found in some isolates of B. intermedia and B. pilosicoli. Some isolates of B. pilosicoli were not susceptible to ampicillin. Additional studies showed that zinc bacitracin, a common feed additive, can increase susceptibility to colonisation with B. pilosicoli. Both tiamulin and lincomycin were shown to be effective in treating infection with B. pilosicoli in adult birds.

Avian intestinal spirochetosis is a disease of poultry caused by the spirochete bacterium Brachyspira, and is characterized by increased mortality, reduced egg production, weight loss, diarrhoea, and fecal staining of egg shells, which leads to downgrading of eggs. The presence of Brachyspira species in Ontario layer chicken flocks and its association with downgrades for dirty eggs was explored. Further, farm interviews were conducted to determine risk factors associated with the presence of Brachyspira species. The prevalence of Brachyspira species was significantly higher in flocks with a higher proportion of dirty eggs (dirty flocks) compared to those with a lower proportion of dirty eggs (clean flocks). Brachyspira pilosicoli was the only pathogenic species detected. Risk factors associated with the presence of Brachyspira species were flock age, barn age, multi-age farms, and housing type. Based on these findings, recommendations were suggested to control the occurrence of Brachyspira species and the associated dirty egg problem. / Animal Health Laboratory

The anaerobic intestinal spirochaete Brachyspira pilosicoli is the causative agent of “intestinal spirochaetosis” (IS), a disease of humans and a number of animal species. IS has been reported in adults and children worldwide but the prevalence in people living in poor hygienic conditions, indigenous populations, homosexual males, and in immunocompromised people is much higher than in other populations. IS is also widespread in pigs and chickens, and causes significant economic impact in the associated industries. To date attempts to develop a vaccine against B. pilosicoli to protect humans and animals have not been successful. In this study a reverse vaccinology approach was used, in which 24 putative open reading frames (ORFs) derived from a partial genome sequence of B. pilosicoli were subjected to in silico and laboratory screening processes to identify potential efficacious vaccine antigens. In silico analysis of the ORFs using a range of bioinformatics algorithms assigned 12 ORF products as periplasmic, outer membrane, or secretory proteins, and these were given a high priority as potential vaccine candidates. The 12 selected ORFs were amplified from a human strain of B. pilosicoli (Wes-B) and cloned. Products from nine ORFS were successfully over-expressed in an Escherichia coli expression system, and then purified using affinity chromatography. In an in vitro immunogenicity trial all the recombinant proteins except for NAV-P27 were strongly recognised in Western immunoblots by a mouse serum raised against B. pilosicoli strain WesB, and by a subset of convalescent sera from pigs naturally and experimentally infected with B. pilosicoli. In an analysis of in vivo immunogenicity, the post-immunisation mouse sera raised against each recombinant protein reacted strongly with each specific proteins, and also recognised the native protein in extracts of B. pilosicoli strain WesB. Sequence analysis of four randomly selected ORFs showed that these were highly conserved amongst the genomes of different human and swine strains of B. pilosicoli. Evaluation of all the data obtained in the reverse vaccinology approach resulted in selection of four ORF products (NAV-P3, NAV-13, NAV-22 and NAV-31) as being potentially protective antigens to be analysed for their further efficacy. These four recombinant proteins were assessed for their efficacy as vaccine components in a mouse model of IS, where the animals were challenged with a human strain of B. pilosicoli. The proteins all induced systemic and local antibody responses, and tended to reduce spirochaete colonisation following experimental infection. These proteins used individually or in combination now have the potential to be further developed into a new vaccine to prevent B. pilosicoli infections.

The work described in this thesis was concerned with identifying the prevalence and risk factors associated with colonisation by the intestinal spirochaete Brachyspira pilosicoli in: humans: long term residents of Perth, Western Australia (WA) and Indonesians either living temporarily in Perth or as long term residents in urban and rural areas of Bali, Indonesia, animals: domestic animals including alpacas, birds, cattle, cats, chickens, dogs, doves, ducks, goats, horses, pigs, and sheep (housed at a wide variety of places around Perth), and a range of wild animals housed in various Zoos and wildlife centres in WA. This study shows that for humans: • Brachyspira pilosicoli was significantly more prevalent in Indonesians of all sub groups, be they temporary residents of Perth (9.4% - 216 faecal samples from 180 individuals), or long term residents of Indonesia (12.6% - 992 faecal samples from 617 individuals) compared with long term residents of Australia living mainly in Perth (0.2% of 766 sampled), even in those with gastrointestinal complaints. This suggests a relationship between a high prevalence of B. pilosicoli and living in Indonesia; • In Bali, B. pilosicoli was significantly more prevalent in the impoverished urban area of Sesetan (20.3-23.4%) where the husbandry of pigs is poor and effluent treatment is non-existent compared to four traditional farming villages (Badung, Karang Suwung, Melinggih, Payangan Desa) (3.3-22.6%). In the latter villages effluent and drainage is better and there is less likely to be contamination of drinking water • There was no significant association between the presence of B. pilosicoli and the presence of clinical symptoms including headaches, abdominal pains, diarrhoea, joint/muscular pain and constipation. • Amongst Indonesians living in Indonesia, there was no significant difference in the prevalence of B. pilosicoli between people with and without contact with animals and between farmers and other occupational groups. • Indonesians visiting Perth who were positive for B. pilosicoli originated from nine cities and five main islands in Indonesia. This suggests that B. pilosicoli is endemic throughout Indonesia. • Strain typing of isolates of B. pilosicoli showed that they were genetically heterogenous and did not show any consistent pattern with respect to geographical location, family of origin or disease status. Isolates from the same individual were sometimes unrelated, suggesting the probability of re-infection with another strain between the samplings. • Some households (~7%) had more than one member positive for B. pilosicoli. Strain analysis suggested transmission between family members, and this could be due to either faecal-oral transmission, or from a common external source, such as contaminated water. • B. pilosicoli was cultured from only 0.2% of Australians. This low prevalence may be a result of little or no exposure to B. pilosicoli due to good personal hygiene and environmental sanitation. • B. pilosicoli strain H1b and H171 that were isolated from healthy Indonesians were able to colonise mice and day-old chickens, and induced clinical signs of pasty faeces in the latter. Histological sections showed mild typhlitis and typical end-on attachment of B. pilosicoli to the caecal epithelial mucosa of the chickens. This finding suggests that the human isolates had pathogenic potential. This study showed that for animals investigated: • Intestinal spirochaetes were cultured from 46.4% (13/28) of bilbies with 14.3% (4/28) positive for B. pilosicoli. Spirochaetes were also cultured from the faeces of two Western Barred bandicoots and one (1.2%) kangaroo. • Intestinal spirochaetes were not isolated from any alpacas, cattle, goats, horses, pigs, and sheep but were detected in 40.5% of ducks, 14.3% of chickens, 14.9% of ostriches and 1.5% of cats. • Few pets that are commonly kept in households (dogs, cats and aviary birds) were colonised, suggesting that they are not an important focus of B. pilosicoli infection in Australia.

Tierärztliche Hochsch., Diss., 2005--Hannover.

Brachyspira hyodysenteriae and Brachyspira pilosicoli are anaerobic intestinal spirochaetes that are the aetiological agents of swine dysentery and intestinal spirochaetosis, respectively. As part of this PhD study the genome sequence of B. hyodysenteriae strain WA1 and a near complete sequence of B. pilosicoli strain 95/1000 were obtained, and subjected to comparative genomic analysis. The B. hyodysenteriae genome consisted of a circular 3.0 Mb chromosome, and a 35,940 bp circular plasmid that has not previously been described. The incomplete genome of B. pilosicoli contained 4 scaffolds. There were 2,652 and 2,297 predicted ORFs in the B. hyodysenteriae and B. pilosicoli strains, respectively. Of the predicted ORFs, more had similarities to proteins of the enteric Clostridium species than they did to proteins of other spirochaetes. Many of these genes were associated with transport and metabolism, and they may have been gradually acquired through horizontal gene transfer in the environment of the large intestine. A reconstruction of central metabolic pathways of the Brachyspira species identified a complete set of coding sequences for glycolysis, gluconeogenesis, a non-oxidative pentose phosphate pathway, nucleotide metabolism and a respiratory electron transport chain. A notable finding was the presence of rfb genes on the B. hyodysenteriae plasmid, and their apparent absence from B. pilosicoli. As these genes are involved in rhamnose biosynthesis it is likely that the composition of the B. hyodysenteriae lipooligosaccharide O-sugars is different from that of B. pilosicoli. O-antigen differences in these related species could be associated with differences in their specific niches, and/or with their disease specificity. Overall, comparison of B. hyodysenteriae and B. pilosicoli protein content and analysis of their central metabolic pathways showed that they have diverged markedly from other spirochaetes in the process of adapting to their habitat in the large intestine. The presence of overlapping genes in the two spirochaetes and in other spirochaete species also was investigated. The number of overlapping genes in the 12 spirochaete genomes examined ranged from 11-45%. Of these, 80% were unidirectional. Overlapping genes were found irregularly distributed within the Brachyspira genomes such that 70-80% of them occurred on the same strand (unidirectional, ->->/<-<-), with 16-28% occurring on opposite DNA strands (divergent, <-->). The remaining 4-6% of overlapping genes were convergent (-><-). The majority of the unidirectional overlap regions were relatively short, with >50% of the total observations overlapping by >4 bp. A small number of overlapping gene-pairs were duplicated within each genome and there were some triplet overlapping genes. Unique orthologous overlapping genes were identified within the various spirochaete genera. Over 75% of the overlapping genes in the Brachyspira species were in the same or related metabolic pathway. This finding suggests that overlapping genes are not only likely to be the result of functional constraints but also are constrained from a metabolomic context. Of the remaining 25% overlapping genes, 50% contained one hypothetical gene with unknown function. In addition, in one of the orthologous overlapping genes in the Brachyspira species, a promoter was shared, indicating the presence of a novel class of overlapping gene operon in these intestinal spirochaetes.

The SmpA outer membrane lipoprotein of B. hyodysenteriae has several characteristics that indicate the potential to protect against swine dysentery (SD). It localises to the outer membrane and antibodies directed against SmpA can prevent the growth of B. hyodysenteriae in vitro. There is some variation observed in the distribution and expression of the SmpA lipoprotein, suggesting that vaccination with SmpA may not provide protection against challenge with a heterologous B. hyodysenteriae strain. This study has characterised the variation at the smpA locus, and in the process has identified a novel gene, smpB. There is very low similarity between smpB and smpA, with the exception of an identical lipoprotein signal sequence. This suggests that SmpB may be translocated to the outer membrane of B. hyodysenteriae in a similar fashion to SmpA. The results described in this thesis indicate that strains of B. hyodysenteriae harbour either smpA or smpB, but not both, explaining the earlier results of Turner et al. (1991). The presumed outer membrane location of SmpB lead to further investigations into its potential to protect mice from infection with B. hyodysenteriae. Swine Dysentery is a inflammatory disease of the swine colon. Therefore it is believed that a mucosal immune response may provide increased protection against challenge. In this study, vaccination of mice with recombinant SmpB elicited high levels of serum antibodies, induced the production of Interleukin-4 producing T lymphocytes and decreased the observed histological effects after challenge with virulent B. hyodysenteriae. In efforts to increase the protected conferred by vaccination with SmpB, recombinant Salmonella typhimurium STM-1 vaccines were created to express SmpB or deliver DNA vaccines encoding SmpB. Vaccination with these recombinant Salmonella vectors did not induce a measurable SmpB specific immune response. Macrophage survival and plasmid stability studies indicated that this was due to instability of the expression plasmids in STM-1. Although SmpB will only ever protect against strains of B. hyodysenteriae harbouring smpB, these results indicate that with further research, SmpB (and SmpA) may contribute to protection from SD. Toxin production is an important aspect of the pathogenesis of many pathogenic bacteria. Vaccination with attenuated toxins is commonly used to prevent disease. In this study, the B. hyodysenteriae â-haemolysin HlyA was used to vaccinate mice to determine the protection induced after challenge. Vaccination of mice with recombinant HlyA induced significant levels of serum antibodies and lowered the observed pathological effects after challenge of vaccinated mice with virulent B. hyodysenteriae. In an attempt to increase the mucosal immune response and therefore the protection afforded after vaccination with HlyA, recombinant S. typhimurium STM-1 strains were created to express HlyA or deliver DNA vaccines encoding HlyA. Similar to the recombinant STM-1 vaccines expressing SmpB, a HlyA specific immune response was not observed by ELISA or ELISPOT analysis. Plasmid stability trials revealed that the inability to induce a detectable HlyA specific immune response by recombinant STM-1 vaccination may be due to ins tability of the plasmids. Outer membrane proteins are often important components of vaccines against bacterial and viral pathogens. Considering the variation observed in the smpA locus in this study resulting in the identification of smpB, further investigation into the distribution and conservation of outer membrane encoding genes in B. hyodysenteriae strains was undertaken. In particular, the blpAEFG, vspABCD and vspEFGH clusters were analysed for their distribution. It was demonstrated that genes that are B. hyodysenteriae specific (vspABCD and vspEFGH) displayed higher levels of polymorphism than those that are distributed amongst non-pathogenic species, such as B. innocens (which contains blpAEFG). This suggests that the variation in the vspABCD and vspEFGH clusters amongst B. hyodysenteriae strains may be a result of the exposure to the host immune system. Further investigation was undertaken by PFGE analysis and 2D-gel electrophoresis, to analyse genomic and proteomic variation at a global level. Although strains of B. hyodyse nteriae produced several different electrophoretic types (ET) upon PFGE analysis, only limited correlation between the PFGE ET, the polymorphisms in vspABCD and vspEFGH and the presence of smpA/smpB were observed. 2D-gel electrophoresis analysis of outer membrane preparations of two B. hyodysenteriae strain revealed several distinct differences in the outer membrane between B. hyodysenteriae strains. The observed differences in the proteins contained in the outer membrane of B. hyodysenteriae is important for vaccine design, as the induction of cross protection between strains of B. hyodysenteriae is essential for a effective vaccine.

Brachyspira species are bacteria that colonize the intestines of some mammalian and avian species with different degrees of pathogenicity. Brachyspira intermedia is a mild pig and bird pathogen with an unknown genomic sequence. In this project, we completed the genome of Brachyspira intermedia PWS/AT and did a comparative genomic analysis between B. intermedia PWS/AT and the already completed genomes of B. hyodysenteriae WA1, B. murdochii 56-150T and B. pilosicoli 95/1000. A table containing 15 classes of unique and shared genes was developed and analyzed in order to gain a better understanding of species-specific traits and clues behind the different degree of pathogenicity. Our result shows that genes are overall poorly annotated and further studies are of great importance for understanding different and shared properties. The largest number of unique features was found in B. intermedia and B. murdochii. B. hyodysenteriae and B. pilosicoli has most likely developed independently towards different biological niches and B. pilosicoli has undergone a major reductive evolution. One plasmid and six prophages were found in B. intermedia, where two of the phages appear to be capable of horizontal gene transfer. Further genome sequencing of more strains will probably increase the understanding of species-specific traits even more.

This thesis aimed to verify the causal association between "B. hampsonii" and the re-emergence of mucohaemorrhagic diarrhea in North American swine farms, to investigate the role of the intestinal microbiome as a predisposing factor for infection, to develop a porcine colon in vitro culture model and to apply this model in investigating early host-pathogen interactions. Two infection trials were conducted to determine the pathogenicity of "B. hampsonii" clade II and clade I. Weanling pigs were divided into control (n=6) and inoculated (n=12) groups. In each trial, pigs were inoculated with "B. hampsonii" clade II (tissue homogenate or pure culture) or clade I (pure culture) or sterile culture media. Animals were monitored for clinical signs of diarrhea and upon observation of bloody diarrhea they were necropsied for characterization of lesions. Fecal shedding of "B. hampsonii" was monitored throughout the trials using culture and quantitative real-time PCR. Pre and post-diarrhea fecal samples from the clade II infection trial were used to study the microbiome response to "B. hampsonii" infection and to determine if pre-inoculation microbiome composition differed between pigs that did or did not develop clinical disease. For in vitro model development, numerous factors associated with explant survivability in culture were investigated to develop a protocol for culture of porcine colon explants. The optimized model was used to study the first 12 hours of "B. hampsonii" clade II interaction with the host using a combination of histopathology and gene expression analysis. Pigs inoculated with "B. hampsonii" clade I (9/11) and clade II (9/12 and 8/12 in the tissue homogenate and pure culture experiments, respectively) developed mucohaemorrhagic diarrhea and colitis within 14 days of inoculation. In all trials, mucohaemorrhagic diarrhea was significantly more common in inoculated pigs than controls. No significant differences in richness, diversity or taxonomic composition distinguished the pre-inoculation microbiomes of affected or unaffected clade II inoculated pigs. After the development of diarrhea, the fecal microbiome of diarrheic pigs was more dense and had a had a lower Bacteroidetes:Firmicutes ratio when compared to inoculated but unaffected or control pigs. Cultured porcine colon explants displayed differentiated epithelium and crypts after 5 days in culture, while expressing GAPDH at a constant rate. For explants to thrive in vitro our results suggested the use of distal spiral colon, processed immediately after euthanasia, and cultured in an oxygen-rich gas mix with air-liquid culture interface in media containing antibiotics and antifungals. Explants exposed to "B. hampsonii" for 12 hours had a greater number of necrotic cells and thicker catarrhal exudate than control explants. Interaction of spirochaetes with the epithelium, necrotic cells and crypts was visible under optical microscopy, and a trend of increased expression of IFN-γ and e-cadherin in inoculated explants relative to control explants was observed. Taken together, results of this thesis demonstrate that "B. hampsonii" causes mucohaemorrhagic diarrhea in pigs and modulates their intestinal microbiome. The development of an in vitro infection model that replicates in vivo features facilitated the observation of the initial events in "B. hampsonii" interaction with the colon. When explants were exposed to "B. hampsonii" similar histological lesions to in vivo were observed. This system provides a powerful model for future studies of the pathogenesis of "B. hampsonii" and other enteric pathogens of pigs.